Not applicable.
This invention pertains to winter oilseed rape (WOSR) plants, more particularly a pair of winter oilseed rape plants, which is particularly suited for the production of hybrid seed. More specifically, the one plant is wherein being male-sterile, due to the presence in its genome of a male-sterility gene, while the other is wherein carrying a fertility-restorer gene, capable of preventing the activity of the male-sterility gene. The pair WOSR plants of the invention combine the ability to form hybrid seed with optimal overall agronomic performance, genetic stability and adaptability to different genetic backgrounds.
All documents cited herein are hereby incorporated herein by reference.
The phenotypic expression of a transgene in a plant is determined both by the structure of the gene itself and by its location in the plant genome. At the same time the presence of the transgene at different locations in the genome will influence the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of a commercially interesting trait in a plant by genetic manipulation can be a lengthy procedure dependent on different factors. The transformation and regeneration of genetically transformed plants are only the first in a series of selection steps that include extensive genetic characterization, breeding, and evaluation in field trials.
Oilseed rape (OSR) (Brassica napus, AACC, 2n=38) is a natural hybrid resulting from the interspecies hybridization between Cole (Brassica oleracea, CC, 2n=18) and Turnip (Brassica campestris, AA, 2n=20). Winter oilseed rape is sown during the last 10 days of August and the first ten days of September and harvested the following July, needing a temperate period for vernalization. The faster growing spring rapes are sown during late March and early April being harvested mid August to September. The main types of OSR grown at present are low and high erucic acid varieties. Double low (00) varieties contain low (typically less than 1%) levels of erucic acids (which humans find hard to digest), and low levels of glucosinolates (which makes the meal by-product indigestible for animals). Current uses for xe2x80x9c00xe2x80x9d varieties include oil for human consumption and high protein meal for animal feed. Industrial uses include feedstocks for pharmaceuticals and hydraulic oils. High erucic acid rape (HEAR) varieties are grown specifically for their erucic acid contentxe2x80x94typically 50-60% of oil. The principal end use of HEAR is to produce erucamide, a xe2x80x9cslip agentxe2x80x9d used in polyethane manufacture. A small portion is used to produce behenyl alcohol, which is added to a waxy crude mineral oil to improve its flow.
Oilseed rape plants are bisexual and typically 60-70% self-pollinated. The production of hybrids and introduction of genetic variation as a basis for selection was traditionally dependent on the adaptation of natural occurring phenomena such as self-incompatibility and cytoplasmic male sterility. Artificial pollination control methods such as manual emasculation or the use of gametocides are not widely applied in OSR breeding due to their limited practicability and high cost respectively.
Transgenic methods have been developed for the production of male or female-sterile plants, which provide interesting alternatives to the traditional techniques.
EP 0,344,029 describes a system for obtaining nuclear male sterility whereby plants are transformed with a male-sterility gene, which comprises, for example a DNA encoding a barnase under the control of a tapetum specific promoter, PTA29, which when incorporated into a plant ensures selective destruction of tapetum cells. Transformation of tobacco and oilseed rape plants with such a chimeric gene resulted in plants in which pollen formation was completely prevented. Mariani et al. (1990) Nature 347:737-741.
To restore fertility in the progeny of a male-sterile plant, a system was developed whereby the male-sterile plant is crossed with a transgenic plant carrying a fertility-restorer gene, which when expressed is capable of inhibiting or preventing the activity of the male-sterility gene (U.S. Pat. No. 5,689,041; U.S. Pat. No. 5,792,929). Such a fertility-restorer gene is placed under the control of a promoter directing expression at least in the cells in which the male-sterility gene is expressed. Mariani et al. ((1992) Nature 357:384-387) demonstrated that the sterility encoded by the pTA29: barnase gene can be restored by the chimeric pTA29: barstar gene in oilseed rape.
Cytochemical and histochemical analysis of anther development of B. napus plants comprising the chimeric pTA29: barnase gene alone or with pTA29: barstar is described by De Block and De Brouwer ((1993) Planta 189:218-225).
Successful transformation of Brassica species has been obtained by a number of methods including Agrobacterium infection (as described for example in EP 0,116,718 and EP 0,270,882), microprojectile bombardment (as described for example by Chen et al. (1994) Theor. Appl. Genet. 88:187-192) and direct DNA uptake (as described for example by De Block et al. (1989) Plant Physiol. 914:694-701; and Poulsen (1996) Plant Breeding 115:209-225.
However, the foregoing documents fail to teach or suggest the present invention.
The present invention relates to a pair of WOSR plants, particularly suited for producing hybrid seed. More particularly, the present invention relates to a first transgenic WOSR plant, or seed, cells or tissues thereof, comprising, integrated into its genome, an expression cassette which comprises a male-sterility gene, and a second transgenic WOSR plant, or seed, cells or tissues thereof, comprising, integrated into its genome, an expression cassette which comprises a fertility restorer gene, and the hybrid seed obtained by the crossing of the first and second plant, which comprises the male-sterility gene and/or the fertility restorer gene integrated into its genome.
In one embodiment, the first WOSR plant or seed, cells or tissues thereof, comprises the expression cassette of pTHW107. In the preferred embodiment of the invention the first WOSR plant or seed, cells or tissues thereof comprise event MS-BN1.
In another embodiment of the invention, the second WOSR plant or seed, cells or tissues thereof, comprises the expression cassette of pTHW1118. In the preferred embodiment of the invention the WOSR plant or seed, cells or tissues thereof comprise event RF-BN1. In a particularly preferred embodiment of the invention the first WOSR plant comprises event MS-BN1 and the second WOSR plant comprises event RF-BN1 and the hybrid seed obtained therefrom comprises event MS-BN1 and/or RF-BN1.
The invention relates to transgenic WOSR seed, or a plant that can be grown from such seed, the genomic DNA of which is wherein one or both of the following characteristics:
a) the genomic DNA is capable of yielding at least two, preferably at least three, more preferably at least four, most preferably five of the sets of restriction fragments selected from the group of:
i) one set of two EcoRI fragments, one with a length of between 2140 and 2450 bp, preferably of about 2266 bp, and one with a length of more than 14 kbp;
ii) one set of two EcoRV fragments wherein one has a length of between 1159 and 1700 bp, preferably of about 1.4 kbp and the other has a length of more than 14 kbp;
iii) one set of two Hpal fragments, one with a length of between 1986 and 2140 bp, preferably with a length of about 1990 bp, and one with a length of between 2140 and 2450 bp, preferably of about 2229 bp;
iv) one set of three AflIII fragments, one with a length of between 514 and 805 bp, preferably with a length of about 522 bp, and one with a length of between 2140 and 2450 bp, preferably about 2250 bp, and one with a length of between 2450 and 2838 bp, preferably of about 2477 bp.;
v) one set of two NdeI fragments, both with a length of between 5077 and 14057 bp, preferably one of about 6500 bp, and one with a length of about 10 kbp;
wherein each of the restriction fragments is capable of hybridizing under standard stringency conditions, with the 3942 bp fragment comprising the PTA29-barnase sequence obtainable by HindIII digestion of the plasmid pTHW107 described herein; and/or
b) the genomic DNA is capable of yielding at least two, preferably at least three, more preferably four of the sets of restriction fragments selected from the group of:
i) one set of three BamHI fragments, wherein one has a length of between 805 and 1099 bp, preferably of about 814 bp, one has a length between 1700 and 1986 bp, preferably of about 1849 bp, one has a length between 2450 and 2838 bp, preferably of about 2607 bp, and one has a length between 5077 and 14057 bp, preferably of about 6500 bp;
ii) one set of four EcoRI fragments, one with a length of between 805 and 1159 bp, preferably of about 1094 bp, one with a length between 1986 and 2450 bp, preferably of about 2149 bp, and two with a length of between 5077 and 14057 bp, preferably one of about 7000 bp, and one with a length of about 10 kbp;
iii) one set of two FcoRV fragments wherein both have a length of between 5077 and 14057 bp, preferably one has a length of about 5.4 kbp and the other has a length of about 8 kbp;
iv) one set of three HindIII fragments, wherein one has a length of between 1700 and 2140 bp, preferably of about 1969 bp, and two have a length between 2450 and 2838 bp, preferably one has a length of about 2565 bp, and one has a length of about 2635 bp;
wherein each of the restriction fragments is capable of hybridizing under standard stringency conditions, with the 2182 bp fragment comprising the PTA29-barstar sequence obtainable by HpaI digestion of the plasmid pTHW118 described herein.
The present invention relates to a the seed of a WOSR plant, or a plant which can be grown from such seed, or cells, or tissues thereof, the genomic DNA of which is wherein one or both of the following characteristics:
a) the genomic DNA is capable of yielding at least two, preferably at least three, for instance at least four, more preferably five of the sets of restriction fragments selected from the group described under a) above comprising the sets of restriction fragments described under a) i), ii), iii), iv), and v) above, whereby the selection can include any combination of i), ii), iii), iv), and v) described under a) above; and/or
b) the genomic DNA is capable of yielding at least two, preferably at least three, most preferably four of the sets of restriction fragments selected from the group described under b) above comprising the sets of restriction fragments described under b) i), ii), iii) and iv) above, whereby the selection can include any combination of i), ii), iii) and iv) described under b) above.
The invention further relates to WOSR seed, or plants grown from such seed, the genomic DNA of which is wherein one or both of the following characteristics:
c) the genomic DNA can be used to amplify a DNA fragment of between 260 and 300 bp, preferably of about 280 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID NO:12 and SEQ ID NO:19 respectively; and/or
d) the genomic DNA can be used to amplify a DNA fragment of between 195 and 235 bp, preferably of about 215 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID NO:23 and SEQ ID NO:41 respectively.
The invention further relates to WOSR seed, or plants grown from such seed, the genomic DNA of which is wherein the characteristics described under a) and c) above and/or the characteristics described under b) and d) above.
The present invention relates to the seed of a WOSR plant, or a plant that can be grown from such seed, the genomic DNA of which is wherein one or both of the following characteristics:
a) the genomic DNA is capable of yielding at least two, preferably at least three, more preferably at least four, most preferably five of the sets of restriction fragments selected from the group of:
i) one set of two EcoRI fragments, one with a length of between 2140 and 2450 bp, preferably of about 2266 bp, and one with a length of more than 14 kbp.
ii) one set of two EcoRV fragments wherein one has a length of between 1159 and 1700 bp, preferably of about 1.4 kbp and the other has a length of more than 14 kbp.
iii) one set of two Hpal fragments, one with a length of between 1986 and 2140 bp, preferably with a length of about 1990 bp, and one with a length of between 2140 and 2450 bp, preferably of about 2229 bp.
iv) one set of three AflIII fragments, one with a length of between 514 and 805 bp, preferably with a length of about 522 bp, one with a length of between 2140 and 2450 bp, preferably about 2250 bp, and one with a length of between 2450 and 2838 bp, preferably of about 2477 bp.
v) one set of two NdeI fragments, both with a length of between 5077 and 14057 bp, preferably one of about 6500 bp, and one with a length of about 10 kbp;
wherein each of the restriction fragments is capable of hybridizing under standard stringency conditions, with the 3942 bp fragment comprising the PTA29-barnase sequence obtainable by HindIII digestion of the plasmid pTHW107 described herein; and/or,
c) the genomic DNA can be used to amplify a DNA fragment of between 260 and 300 bp, preferably of about 280 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID NO:12 and SEQ ID NO:19 respectively.
The present invention relates to a the seed of a WOSR plant, preferably a male-sterile plant, or a plant which can be grown from such seed, or cells, or tissues thereof, the genomic DNA of which is wherein it is capable of yielding at least two, preferably at least three, more preferably five of the sets of restriction fragments selected from the group described above comprising the sets of restriction fragments described under i), ii), iii), iv), and v) above, whereby the selection can include any combination of i), ii), iii), iv), and v) described above.
The present invention further relates to the seed of a WOSR plant, or a plant grown from such seed, the genomic DNA of which is wherein one or both of the following characteristics:
b) the genomic DNA is capable of yielding at least two, preferably at least three, more preferably four of the restriction fragments or sets of restriction fragments selected from the group of.
i) one set of three BamHI fragments, wherein one has a length of between 805 and 1099 bp, preferably of about 814 bp, one has a length between 1700 and 1986 bp, preferably of about 1849 bp, one has a length between 2450 and 2838 bp, preferably of about 2607 bp, and one has a length between 5077 and 14057 bp, preferably of about 6500 bp;
ii) one set of four EcoRI fragments, one with a length of between 805 and 1159 bp, preferably of about 1094 bp, one with a length between 1986 and 2450 bp, preferably of about 2149 bp, and two with a length of between 5077 and 14057 bp, preferably one of about 7000 bp, and one with a length of about 10 kbp;
iii) one set of two EcoRV fragments wherein both have a length of between 5077 and 14057 bp, preferably one has a length of about 5.4 kbp and the other has a length of about 8 kbp;
iv) one set of three HindIII fragments, wherein one has a length of between 1700 and 2140 bp, preferably of about 1969 bp, and two have a length between 2450 and 2838 bp, preferably one has a length of about 2565 bp, and one has a length of about 2635 bp;
wherein each of the restriction fragments is capable of hybridizing under standard stringency conditions, with the 2182 bp fragment comprising the PTA29-barstar sequence obtainable by HpaI digestion of the plasmid pTHW118 described herein; and/or
d) the genomic DNA can be used to amplify a DNA fragment of between 195 and 235 bp, preferably of about 215 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID NO:23 and SEQ ID NO:41 respectively.
The present invention relates to the seed of a WOSR plant, preferably a fertility restorer plant, or a plant which can be grown from such seed, or cells, or tissues thereof, the genomic DNA of which is capable of yielding at least two, preferably at least three, most preferably four of the sets of restriction fragments selected from the group described above comprising the sets of restriction fragments described under b) i), ii), iii) and iv) above, whereby the selection can include any combination of i), ii), iii) and iv) described above.
The present invention relates to transgenic WOSR plants, cells, tissues or seeds that preferably contain both of the characteristics described under b) and/or d) above, respectively.
The invention further relates to transgenic, preferably hybrid fertility restored WOSR plants, cells, tissues or seeds obtained from the crossing of the male-sterile plant with the fertility restorer plant of the invention wherein the respective characteristics described above, whereby the fertility restored plants, cells tissues or seeds are wherein both the molecular characteristics of the male-sterile and those of the fertility restorer WOSR plant described above. The invention further relates to transgenic, preferably hybrid WOSR plants, cells, tissues or seeds obtained from the crossing of the male-sterile plant with the fertility restorer plant of the invention wherein the molecular characteristics described above, whereby the hybrid plants, cells tissues or seeds are wherein the molecular characteristics of the fertility restorer WOSR plant described above.
The invention also relates to the seed deposited at the ATCC under accession number PTA-730, a plant which is grown from this seed, and cells or tissues from a plant grown from this seed. The invention further relates to plants obtainable by propagation of, and/or breeding with a WOSR plant grown from the seed deposited at the ATCC under accession number PTA-730.
The invention further relates to a process for producing hybrid WOSR seed, which comprises, crossing the male-sterile WOSR plant of the present invention with the fertility-restorer WOSR plant of the invention.
The invention further relates to a WOSR plant, plant cell, plant tissue or seed, which comprises a recombinant DNA comprising at least one transgene, integrated into a part of the chromosomal DNA wherein the sequence of SEQ ID NO:22 and/or a recombinant DNA comprising at least one transgene, integrated into a part of the chromosomal DNA wherein the sequence of SEQ ID NO:34.
The invention further provides a process for producing a transgenic cell of a WOSR plant or a plant obtained therefrom, which comprises inserting a recombinant DNA molecule into a part of the chromosomal DNA of an WOSR cell wherein the sequence of SEQ ID NO:22 and, optionally, regenerating a WOSR plant from the transformed WOSR cell.
The invention further provides a process for producing a transgenic cell of a WOSR plant or a plant obtained therefrom, which comprises inserting a recombinant DNA molecule into a part of the chromosomal DNA of an WOSR cell wherein the sequence of SEQ ID NO:34 and, optionally, regenerating a WOSR plant from the transformed WOSR cell.
The invention further relates to a method for identifying a transgenic plant, or cells or tissues thereof, comprising the elite event MS-BN1 of the invention, which method comprises establishing one or both of the following characteristics of the genomic DNA of the transgenic plant, or its cells or tissues:
a) the genomic DNA is capable of yielding at least two, preferably at least three, more preferably at least four, most preferably five of the sets of restriction fragments selected from the group of:
i) one set of two EcoRI fragments, one with a length of between 2140 and 2450 bp, preferably of about 2266 bp, and one with a length of more than 14kbp;
ii) one set of two EcoRV fragments wherein one has a length of between 1159 and 1700 bp, preferably of about 1.4 kbp and the other has a length of more than 14 kbp;
iii) one set of two Hpal fragments, one with a length of between 1986 and 2140 bp, preferably with a length of about 1990 bp, and one with a length of between 2140 and 2450 bp, preferably of about 2229 bp;
iv) one set of three AflIII fragments, one with a length of between 514 and 805 bp, preferably with a length of about 522 bp, one with a length of between 2140 and 2450 bp, preferably about 2250 bp, and one with a length of between 2450 and 2838 bp, preferably of about 2477 bp; and
v) one set of two NdeI fragments, both with a length of between 5077 and 14057 bp, preferably one of about 6500 bp, and one with a length of about 10 kbp;
wherein each of the restriction fragments is capable of hybridizing under standard stringency conditions, with the 3942 bp fragment comprising the PTA29-barnase sequence obtainable by HindIII digestion of the plasmid pTHW107 described herein; and/or
c) the genomic DNA can be used to amplify a DNA fragment of between 260 and 300 bp, preferably of about 280 bp, according to the PCR Identification Protocol described herein with two primers identifying the elite event having the nucleotide sequence of SEQ ID NO:12 and SEQ ID NO:19 respectively.
The invention further relates to a method for identifying a transgenic plant, or cells or tissues thereof, comprising the elite event RF-BN1 of the invention, which method comprises establishing one or both of the following characteristics of the genomic DNA of the transgenic plant, or its cells or tissues:
b) the genomic DNA is capable of yielding at least two, preferably at least three, more preferably four of the restriction fragments or sets of restriction fragments selected from the group of:
i) one set of three BamHI fragments, wherein one has a length of between 805 and 1099 bp, preferably of about 814 bp, one has a length between 1700 and 1986 bp, preferably of about 1849 bp, one has a length between 2450 and 2838 bp, preferably of about 2607 bp, and one has a length between 5077 and 14057 bp, preferably of about 6500 bp;
ii) one set of four EcoRI fragments, one with a length of between 805 and 1159 bp, preferably of about 1094 bp, one with a length between 1986 and 2450 bp, preferably of about 2149 bp, and two with a length of between 5077 and 14057 bp, preferably one of about 7000 bp, and one with a length of about 10 kbp;
iii) one set of two EcoRV fragments wherein both have a length of between 5077 and 14057 bp, preferably one has a length of about 5.4 kbp and the other has a length of about 8 kbp;
iv) one set of three HindIII fragments, wherein one has a length of between 1700 and 2140 bp, preferably of about 1969 bp, and two have a length between 2450 and 2838 bp, preferably one has a length of about 2565 bp, and one has a length of about 2635 bp;
wherein each of the restriction fragments is capable of hybridizing under standard stringency conditions, with the 2182 bp fragment comprising the PTA29-barstar sequence obtainable by HpaI digestion of the plasmid pTHW118 described herein, and/or
d) the genomic DNA can be used to amplify a DNA fragment of between 195 and 235 bp, preferably of about 215 bp, using the PCR identification protocol described herein with two primers identifying the elite event having the nucleotide sequence of SEQ ID NO:23 and SEQ ID NO:41 respectively.
The invention also relates to a kit for identifying the plants comprising elite event MS-BN1 of the present invention, said kit comprising the PCR probes having the nucleotide sequence of SEQ ID NO:12 and SEQ ID NO:19.
The invention further relates to a kit for identifying the plants comprising elite event RF-BN1 of the present invention, said kit comprising the PCR probes having the nucleotide sequence of SEQ ID NO:23 and SEQ ID NO:41.
The invention also relates to a kit for identifying elite event MS-BN1 and/or RF-BN1 in biological samples, which kit comprises at least one specific primer or probe having a sequence which corresponds (or is complementary to) a sequence having between 80% and 100% sequence identity with a specific region of MS-BN1 and/or at least one specific primer or probe having a sequence which corresponds (or is complementary to) a sequence having between 80% and 100% sequence identity with a specific region of RF-BN1. Preferably the sequence of the probe corresponds to a specific region comprising part of the 5xe2x80x2 or 3xe2x80x2 flanking region of MS-BN1 and/or RF-BN1. Most preferably the specific probe has (or is complementary to) a sequence having between 80% and 100% sequence identity to the plant DNA sequence within SEQ ID NO:36 or SEQ ID NO:38 for MS-BN1 or to the plant DNA sequence within SEQ ID NO:39 or SEQ ID NO:40 for RF-BN1.
Preferably the kit of the invention comprises, in addition to a primer which specifically recognizes the 5xe2x80x2 or 3xe2x80x2 flanking region of MS-BN1 and/or RF-BN1, a second primer which specifically recognizes a sequence within the foreign DNA of MS-BN1 and/or RF-BN1, for use in a PCR identification protocol. Preferably, the kit of the invention comprises two (or more) specific primers, one of which recognizes a sequence within the 3xe2x80x2flanking region of MS-BN1 and/or RF-BN1, most preferably a sequence within the plant DNA region of SEQ ID NO:36 or SEQ ID NO:38 for MS-BN1 or to the plant DNA sequence of SEQ ID NO:39 or SEQ ID NO:40 for RF-BN1, and another which recognizes a sequence within the foreign DNA of MS-BN1 and/or RF-BN1 respectively. Especially preferably, the primer recognizing the plant DNA sequence within 5xe2x80x2 flanking region of MS-BN1 comprises the nucleotide sequence of SEQ ID NO:19. Particularly, the primer recognizing the plant DNA sequence within 5xe2x80x2flanking region of MS-BN1 comprises the nucleotide sequence of SEQ ID NO:19 and the primer recognizing the foreign DNA of MS-BN1 comprises the nucleotide sequence of SEQ ID NO:12 described herein. Especially preferably, the primer recognizing the plant DNA sequence within 5xe2x80x2flanking region of RF-BN1 comprises the nucleotide sequence of SEQ ID NO:41. Particularly, the primer recognizing the plant DNA sequence within 5xe2x80x2flanking region of MS-BN1 comprises the nucleotide sequence of SEQ ID NO:41 and the primer recognizing the foreign DNA of RF-BN1 comprises the nucleotide sequence of SEQ ID NO:23 described herein.
The methods and kits encompassed by the present invention can be used for different purposes such as, but not limited to the following: to identify MS-BN1 and/or RF-BN1 in plants, plant material or in products such as, but not limited to food or feed products (fresh or processed) comprising or derived from plant material; additionally or alternatively, the methods and kits of the present invention can be used to identify transgenic plant material for purposes of segregation between transgenic and non-transgenic material; additionally or alternatively, the methods and kits of the present invention can be used to determine the quality (i.e. percentage pure material) of plant material comprising MS-BN1 and/or RF-BN1.
It will be understood that particular embodiments of the invention are described by the dependent claims cited herein.